The applicant previously has isolated transverse tubules and triads from skeletal muscle and evaluated the biochemical and physiological properties of the organelles. The current proposal is directed towards a detailed morphology, structure and biochemistry of the triadic junction region of skeletal muscle. The ultimate goal is to understand the mechanism of excitation-contraction coupling. The methods which will be employed will be to extract, identify and isolate proteins from a preparation of intact triads. One protein has molecular weight 1.2 X one million and is considered to be the spanning protein of the triad. Monoclonal antibody has been directed against this protein. It will be employed to assist in purifying the protein and to localize the protein by microscopy and electron microscopy in muscle and other tissues. The spanning protein will be employed in cross linking experiments with triadic vesicles in order to determine which proteins it interacts with in the triad. The protein will be examined by electron microscopy to determine its structure. The identity of proteins which anchor the spanning protein to the membranes of the T-tubule and terminal cisternae and the nature of the interaction of calsequestrin with the triad will be elucidated. This work will be carried out employing cross linking agents and by extraction and isolation of proteins. Antibodies will be prepared and employed as described above. We have identified a protein which promotes the formation of the triad junction from T-tubules and terminal cisternae as glyceraldehyde phosphate dehydrogenase. This protein binds to calsequestrin. We have recently discovered that the enzyme is phosphorylated by ATP and that it promotes the phosphorylation of triadic proteins. The mechanism of this control of protein phosphorylation will be investigated. The goal is to determine how the action is triggered physiologically, what the intermediates are, what the kinetics of phosphorylation are and what the physiological consequences are. The role of phosphorylation in influencing Ca2+ accumulation and release in sarcoplasmic reticulum and T-tubules will be evaluated.
